Stable Membrane Potential Research Articles

Triphenyltin chloride exposure inhibits meiotic maturation of mouse oocytes by disrupting cytoskeleton assembly and cell cycle progression

Triphenyltin chloride (TPTCL) is widely used in various industrial and agricultural applications. This study aimed to elucidate the mechanisms underlying the toxicological effects of TPTCL on oocytes. The obtained findings revealed that TPTCL exposure reduced polar body extrusion (PBE) and induced meiotic arrest. Mechanistically, TPTCL disrupted meiotic spindle assembly and chromosome alignment. Further analysis indicated a significant decrease in p-MAPK expression, and disturbances in the localization of Pericentrin and p-Aurora A in TPTCL exposed oocytes, which suggesting impaired microtubule organizing center (MTOC)function. Moreover, TPTCL exposure enhance microtubule acetylation and microtubule instability. Therefore, the spindle assembly checkpoint (SAC) remained activated, and the activity of the anaphase-promoting complex (APC) was inhibited, thereby preventing oocytes from progressing into the entering anaphase I (AI) stage. TPTCL exposure also augmented the actin filaments in the cytoplasm. Notably, mitochondrial function appeared unaffected by TPTCL, as evidenced indicated by stable mitochondrial membrane potential and ATP content. Furthermore, TPTCL treatment altered H3K27me2, H3K27me3 and H3K9me3 levels, suggesting changes in epigenetic modifications in oocytes. Taken together, our results suggest that TPTCL disrupts cytoskeleton assembly, continuously activates SAC, inhibits APC activity, and blocks meiotic progression, ultimately impair oocyte maturation.

Transmembrane potential, an indicator in situ reporting cellular senescence and stress response in plant tissues

BackgroundPlant cells usually sustain a stable membrane potential due to influx and/or efflux of charged ions across plasma membrane. With the growth and development of plants, different tissues and cells undergo systemic or local programmed decline. Whether the membrane potential of plasma membrane could report senescence signal of plant tissues and cells is unclear.ResultsWe applied a maneuverable transmembrane potential (TMP) detection method with patch-clamp setup to examine the senescence signal of leaf tissue cells in situ over the whole life cycle in Arabidopsis thaliana. The data showed that the TMPs of plant tissues and cells were varied at different growth stages, and the change of TMP was higher at the vegetative growth stage than at the reproductive stage of plant growth. The distinct change of TMP was detectable between the normal and the senescent tissues and cells in several plant species. Moreover, diverse abiotic stimuli, such as heat stress, hyperpolarized the TMP in a short time, followed by depolarized membrane potential with the senescence occurring. We further examined the TMP of plant chloroplasts, which also indicates the senescence signal in organelles.ConclusionsThis convenient TMP detection method can report the senescence signal of plant tissues and cells, and can also indicate the potential of plant tolerance to environmental stress.

Mitochondrial bioenergetic, oxidative stress and burrowing responses in earthworm exposed to roxarsone in soil

The eco-risk of roxarsone (ROX) was evaluated using multiple responses of earthworm biomarkers under different ROX concentrations for 28 d. With the increasing total arsenic accumulation (TAs-E), biological responses in earthworm generally showed a two-stage changes of homeostasis dysregulation and dose-dependent alterations. At the early periods, ROX stress increased the reactive oxygen species (ROS) and lipid peroxidation (LPO) in a similar manner, and apparently disrupted mitochondrial calcium ([Ca2+]m). But earthworms regulated their mitochondrial and redox homeostasis through stable mitochondrial membrane potential (MMP) and increase of ATP level, superoxide dismutase (SOD) and catalase (CAT). After 14 d, the positively correlated mitochondrial effects of ROS, [Ca2+]m, MMP and ATP were related to the behavioral inhibition of burrow length, depth and reuse rate as well as antioxidant up-regulation of Nrf2, HO-1, sod1 and cat. These results contributed possible biomarkers from the dose-dependent relationship between mitochondrial, antioxidant and behavioral responses. Multiple biological detection in earthworms can better reflect the sub-chronic ecotoxicity of phenylarsenic pollutants in soil.

LATS2 Deletion Attenuates Myocardial Ischemia-Reperfusion Injury by Promoting Mitochondrial Biogenesis.

Reperfusion therapy is the most effective treatment for acute myocardial infarction, but it can damage cardiomyocytes through a mechanism known as myocardial ischemia/reperfusion injury (MIRI). In this study, we investigated whether the large tumor suppressor kinase 2 (LATS2) contributes to the development of myocardial MIRI by disrupting mitochondrial biogenesis. Our in vitro data demonstrate that cardiomyocyte viability was reduced and apoptosis was increased in response to hypoxia/reoxygenation (H/R) injury. However, suppression of LATS2 by shRNA sustained cardiomyocyte viability by maintaining mitochondrial function. Compared to H/R-treated control cardiomyocytes, cardiomyocytes transfected with LATS2 shRNA exhibited increased mitochondrial respiration, improved mitochondrial ATP generation, and more stable mitochondrial membrane potential. LATS2 suppression increased cardiomyocyte viability and mitochondrial biogenesis in a manner dependent on PGC1α, a key regulator of mitochondrial metabolism. These results identify LATS2 as a new inducer of mitochondrial damage and myocardial MIRI and suggest that approaches targeting LATS2 or mitochondrial biogenesis may be beneficial in the clinical management of cardiac MIRI.

MCL Cells in Tumor Microenvironment Impair T-Cell Metabolic Fitness and Effector Function

Background: Mantle cell lymphoma (MCL) is an aggressive, incurable B cell malignancy. Major advances have been achieved with the development of clinically effective targeted agents including BTK and BCL-2 inhibitors. However, patients often relapse after these treatments. The emerging immune therapies combining immune checkpoint blockade and adoptive T-cell transfer are becoming increasingly desirable, but patient responses are heterogeneous, with a significant rate of failure. Tumor microenvironment (TME) plays an important role by inducing immune checkpoint and immune suppressive signaling or creating a metabolic barrier to antitumor immunity, particularly effector T-cells. To evade immune defenses, tumor and stromal cells cooperate and tactically express inhibitory ligands, such as PD-L1, while upregulating immune checkpoint receptors on effector T-cells and CAR T cells, resulting in negative signaling that leads to T-cell exhaustion, one of the major challenges in CAR T-cell therapy. Limited nutrient accessibility and a hostile immunosuppressive TME posts a strong barrier to effective immune therapies, with T-cell immunity being mostly affected. Impaired metabolism may also inhibit the function of effector T-cells while promoting suppressive activity of regulatory T-cells. Results: Our recent transcriptomic analysis of primary MCL clinical specimens comprised of microenvironment cell populations, revealed that genes related to immune function were dysregulated, particularly the genes responsible for IFNG-mediated anti-tumor immunity. Proliferation in response to TCR stimulation is an important measurement for T-cell effector function and potency. Reduced proliferation upon T-cell activation denotes T-cell dysfunction. Indeed, T-cells isolated from MCL specimens especially CAR T non-response patients exhibit diminished cell division upon stimulation with anti-CD3/CD28 beads (25-75% less expansion). To recapitulate the interplay between T-cells and TME, MCL cells were co-cultured with the non-MCL fraction from the purification, and T-cells were then phenotypically profiled. Intriguingly, MCL-derived T-cells mostly displayed exhaustion phenotypes, indicated by decreased expression of T-cell activation markers (CD25, CD28, CD38, and CD71), but increased expression of exhaustion and senescence molecules (CTLA4, LAG3 and PD-1). Moreover, MCL-derived T cells failed to induce the expression of cytokine, such as INF-γ (<1-1.8 vs. 7.1 fold induction) and TNF-α (<1-1.3 vs. 10.2 fold induction), upon TCR stimulation, in contrast to T-cells derived from healthy donors, implicative of reduced cytotoxicity towards tumor cells. The results led us to further attest our hypothesis that MCL cells induce T-cell exhaustion by modulating the expression of inhibitory molecules or immune checkpoint molecules. Metabolic fitness is crucial for T-cell survival and effector function upon activation. A stable mitochondria membrane potential (ΔΨm) is required to maintain metabolic fitness of effector T-cells. The rate of glucose uptake is considered a prominent indicator of T-cell activation. However, MCL derived T-cells displayed remarkable decreased glucose uptake (fold MFI <1-1.21 vs. 1.95), mitochondria mass (p = 0.011) and ΔΨm (fold MFI <1-1.28 vs. 1.9), compared to those from healthy donors, indicating T-cell mitochondrial biogenesis and fitness are impaired in the MCL cell dominated TME. To assess the anti-tumor immunity of T-cells in MCL, we also examined the cytotoxicity of MCL-derived tumor specific cytotoxic T-lymphocytes (CTL) and observed compromised effector activity compared to that from healthy donors. Conclusion: We presented evidence that MCL cancer cells suppress immune cell function by upregulating immunosuppressive signaling and forging a metabolic barrier to effector T-cells and immunotherapy. Targeting the MCL TME while enhancing T-cell metabolic fitness would dramatically advance current immunotherapy. Despite the success of checkpoint blockade and adoptive cell transfer therapy, complimentary approaches to overcome the metabolic barrier of immunosuppressive TME holds promise to dramatically enhance the efficacy of immunotherapies. Further, identification of the precise pathways and targets that determine T-cell metabolic fitness would facilitate developing new approaches to bolster current immunotherapy. Disclosures Wang: AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Acerta Pharma: Research Funding; Oncternal: Consultancy, Research Funding; Guidepoint Global: Consultancy; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Dava Oncology: Honoraria; Molecular Templates: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Lu Daopei Medical Group: Honoraria; InnoCare: Consultancy; OncLive: Honoraria; Verastem: Research Funding; VelosBio: Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; MoreHealth: Consultancy; Targeted Oncology: Honoraria.

Monitoring the Functionality and Stress Response of Yeast Cells Using Flow Cytometry.

Throughout fermentation, yeast faces continuously changing medium conditions and reacts by adapting its metabolism. The adaptation is a critical process and is dependent on the accurate functioning of the cell. A stable membrane potential, which is, among other roles, responsible for protecting the yeast from low pH, is an important attribute for evaluating functionality. Other factors are storage products such as glycogen, trehalose, and neutral lipids, as well as mitochondrial activity and the integrity of the DNA. These parameters can be complemented by the analysis of viability, cell cycle, intracellular pH, and reactive oxygen species in the cell. The correlation of all these factors provides valuable information for evaluating the performance of a yeast population during fermentation. In order to demonstrate the analytical capabilities of flow cytometry, a Saccharomyces cerevisiae yeast strain was observed in a modified growth medium for 384 h (16 days). The results confirm observations made with other methods and reports from the literature. However, with flow cytometry, it is possible to gain deeper insight into stress response and adaptation behavior of yeast at a cellular level. The causality from the formation of oxygen-radicals to cell death, for example, can be shown, as well as the dependency of the intracellular pH on the stability of the membrane. The proposed bio-monitoring system has the potential to provide applicable information as a process control tool for wineries.

Artificial K+ Channels Formed by Pillararene-Cyclodextrin Hybrid Molecules: Tuning Cation Selectivity and Generating Membrane Potential.

A class of artificial K+ channels formed by pillararene-cyclodextrin hybrid molecules have been designed and synthesized. These channels efficiently inserted into lipid bilayers and displayed high selectivity for K+ over Na+ in fluorescence and electrophysiological experiments. The cation transport selectivity of the artificial channels is tunable by varying the length of the linkers between pillararene and cyclodexrin. The shortest channel showed specific transmembrane transport preference for K+ over all alkali metal ions (selective sequence: K+ > Cs+ > Rb+ > Na+ > Li+ ), and is rarely observed for artificial K+ channels. The high selectivity of this artificial channel for K+ over Na+ ensures specific transmembrane translocation of K+ , and generated stable membrane potential across lipid bilayers.

Artificial K+Channels Formed by Pillararene‐Cyclodextrin Hybrid Molecules: Tuning Cation Selectivity and Generating Membrane Potential

AbstractA class of artificial K+channels formed by pillararene‐cyclodextrin hybrid molecules have been designed and synthesized. These channels efficiently inserted into lipid bilayers and displayed high selectivity for K+over Na+in fluorescence and electrophysiological experiments. The cation transport selectivity of the artificial channels is tunable by varying the length of the linkers between pillararene and cyclodexrin. The shortest channel showed specific transmembrane transport preference for K+over all alkali metal ions (selective sequence: K+> Cs+> Rb+> Na+> Li+), and is rarely observed for artificial K+channels. The high selectivity of this artificial channel for K+over Na+ensures specific transmembrane translocation of K+, and generated stable membrane potential across lipid bilayers.

The Role of Na+ and K+ Transporters in Salt Stress Adaptation in Glycophytes.

Ionic stress is one of the most important components of salinity and is brought about by excess Na+ accumulation, especially in the aerial parts of plants. Since Na+ interferes with K+ homeostasis, and especially given its involvement in numerous metabolic processes, maintaining a balanced cytosolic Na+/K+ ratio has become a key salinity tolerance mechanism. Achieving this homeostatic balance requires the activity of Na+ and K+ transporters and/or channels. The mechanism of Na+ and K+ uptake and translocation in glycophytes and halophytes is essentially the same, but glycophytes are more susceptible to ionic stress than halophytes. The transport mechanisms involve Na+ and/or K+ transporters and channels as well as non-selective cation channels. Thus, the question arises of whether the difference in salt tolerance between glycophytes and halophytes could be the result of differences in the proteins or in the expression of genes coding the transporters. The aim of this review is to seek answers to this question by examining the role of major Na+ and K+ transporters and channels in Na+ and K+ uptake, translocation and intracellular homeostasis in glycophytes. It turns out that these transporters and channels are equally important for the adaptation of glycophytes as they are for halophytes, but differential gene expression, structural differences in the proteins (single nucleotide substitutions, impacting affinity) and post-translational modifications (phosphorylation) account for the differences in their activity and hence the differences in tolerance between the two groups. Furthermore, lack of the ability to maintain stable plasma membrane (PM) potentials following Na+-induced depolarization is also crucial for salt stress tolerance. This stable membrane potential is sustained by the activity of Na+/H+ antiporters such as SOS1 at the PM. Moreover, novel regulators of Na+ and K+ transport pathways including the Nax1 and Nax2 loci regulation of SOS1 expression and activity in the stele, and haem oxygenase involvement in stabilizing membrane potential by activating H+-ATPase activity, favorable for K+ uptake through HAK/AKT1, have been shown and are discussed.

High Osmolarity Environments Activate the Mitochondrial Alternative Oxidase in Debaryomyces Hansenii.

The oleaginous yeast Debaryomyces hansenii is a good model to understand molecular mechanisms involved in halotolerance because of its impressive ability to survive under a wide range of salt concentrations. Several cellular adaptations are implicated in this response, including the presence of a cyanide-insensitive ubiquinol oxidase (Aox). This protein, which is present in several taxonomical orders, has been related to different stress responses. However, little is known about its role in mitochondria during transitions from low to high saline environments. In this report, we analyze the effects of Aox in shifts from low to high salt concentrations in the culture media. At early stages of a salt insult, we observed that this protein prevents the overflow of electrons on the mitochondrial respiratory chain, thus, decreasing the production of reactive oxygen species. Interestingly, in the presence of high osmolite concentrations, Aox activity is able to sustain a stable membrane potential when coupled to complex I, despite a compromised cytochrome pathway. Taken together, our results suggest that under high osmolarity conditions Aox plays a critical role regulating mitochondrial physiology.

TERT alleviates irradiation-induced late rectal injury by reducing hypoxia-induced ROS levels through the activation of NF-κB and autophagy.

The hypoxic microenvironment which is present following irradiation has been proven to promote radiation-induced injury to normal tissues. Previous studies have demonstrated that telomerase reverse transcriptase (TERT) is regulated by hypoxia, and that it plays a protective role in the process of wound repair. However, its effects on radiation-induced injury remain unclear. In this study, we examined the effects of human TERT on irradiation-induced late rectal injury in fibroblasts under hypoxic conditions. We also performed in vivo experiments. The rectums of 5-week-old female C57BL/6N mice were irradiated locally with a single dose of 25 Gy. We then examined the fibrotic changes using hematoxylin and eosin staining, and Masson's staining. The expression of hypoxia inducible factor-1α (HIF-1α) and TERT was analyzed by immunohistochemistry. In in vitro experiments, apoptosis, reactive oxygen species (ROS) production and the autophagy level induced by exposure to hypoxia were assayed in fibroblasts. The association between TERT, nuclear factor-κB (NF-κB) and the autophagy level was examined by western blot analysis. The antioxidant effects of TERT were examined on the basis of the ratio of glutathione to glutathione disulfide (GSH/GSSG) and mitochondrial membrane potential. Rectal fibrosis was induced significantly at 12 weeks following irradiation. The HIF-1α and TERT expression levels increased in the fibrotic region. The TERT-overexpressing fibroblasts (transfected with an hTERT-expressing lentiviral vector) exhibited reduced apoptosis, reduced ROS production, a higher autophagy level, a higher GSH/GSSG ratio and stable mitochondrial membrane potential compared with the fibroblasts in which TERT had been silenced by siRNA. NF-κB was activated by TERT, and the inhibition of TERT reduced the autophagy level in the fibroblasts. These results demonstrate that TERT decreases cellular ROS production, while maintaining mitochondrial function and protecting the cells from hypoxia-induced apoptosis, which may thus attenuate the effects of irradiation-induced hypoxia on rectal injury following irradiation.

Genetic Deletion of TREK-1 or TWIK-1/TREK-1 Potassium Channels does not Alter the Basic Electrophysiological Properties of Mature Hippocampal Astrocytes In Situ.

We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K+ channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (VM) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K+ channels remains elusive. TREK-1 two-pore domain K+ channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of heterodimer TWIK-1/TREK-1 channel to the membrane and contributing to astrocyte passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K+ channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, VM, or membrane input resistance (Rin) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K+ channels may shed light on this long-standing and important question in astrocyte physiology.

Freshly dissociated mature hippocampal astrocytes exhibit passive membrane conductance and low membrane resistance similarly to syncytial coupled astrocytes.

Mature astrocytes exhibit a linear current-to-voltage K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.

Rapid developmental emergence of stable depolarization during wakefulness by inhibitory balancing of cortical network excitability.

The ability to generate behaviorally appropriate cortical network states is central to sensory perception and plasticity, but little is known about the timing and mechanisms of their development. I paired intracellular and extracellular recordings in the visual cortex of awake infant rats to determine the synaptic and circuit mechanisms regulating the development of a key network state, the persistent and stable subthreshold membrane potential (Vm) depolarization associated with wakefulness/alertness in cortical networks, called the "desynchronized" or "activated" state. Current-clamp recordings reveal that the desynchronized state is absent during the first 2 postnatal weeks, despite behavioral wakefulness. During this period, Vm remains at the resting membrane potential >80% of the time, regardless of behavioral state. Vm dynamics during spontaneous or light-evoked activity were highly variable, contained long-duration supratheshold plateau potentials, and high spike probability, suggesting an unstable and hyperexcitable early cortical network. Voltage-clamp recordings reveal that effective feedforward inhibition is absent at these early ages despite the presence of feedback inhibition. Stable membrane depolarization during wakefulness finally emerges 1-2 d before eye opening and is statistically indistinguishable from that in adults within days. Reduced cortical excitability, fast feedforward inhibition, and the slow cortical oscillation appear simultaneously with stable depolarization, suggesting that an absence of inhibitory balance during early development prevents the expression of the active state and hence a normal wakeful state in early cortex. These observations identify feedforward inhibition as a potential key regulator of cortical network activity development.

Two Types of Burst Firing in Gonadotrophin‐Releasing Hormone Neurones

Gonadotrophin-releasing hormone (GnRH) neurones fire spontaneous bursts of action potentials, although little is understood about the underlying mechanisms. In the present study, we report evidence for two types of bursting/oscillation driven by different mechanisms. Properties of these different types are clarified using mathematical modelling and a recently developed active-phase/silent-phase correlation technique. The first type of GnRH neurone (1-2%) exhibits slow (∼0.05 Hz) spontaneous oscillations in membrane potential. Action potential bursts are often observed during oscillation depolarisation, although some oscillations were entirely subthreshold. Oscillations persist after blockade of fast sodium channels with tetrodotoxin (TTX) and blocking receptors for ionotropic fast synaptic transmission, indicating that they are intrinsically generated. In the second type of GnRH neurone, bursts were irregular and TTX caused a stable membrane potential. The two types of bursting cells exhibited distinct active-phase/silent-phase correlation patterns, which is suggestive of distinct mechanisms underlying the rhythms. Further studies of type 1 oscillating cells revealed that the oscillation period was not affected by current or voltage steps, although amplitude was sometimes damped. Oestradiol, an important feedback regulator of GnRH neuronal activity, acutely and markedly altered oscillations, specifically depolarising the oscillation nadir and initiating or increasing firing. Blocking calcium-activated potassium channels, which are rapidly reduced by oestradiol, had a similar effect on oscillations. Kisspeptin, a potent activator of GnRH neurones, translated the oscillation to more depolarised potentials, without altering period or amplitude. These data show that there are at least two distinct types of GnRH neurone bursting patterns with different underlying mechanisms.

Function of proton channels in lung epithelia

The properties of the voltage-dependent H(+) channel have been studied in lung epithelial cells for many years, and recently HVCN1 mRNA expression has been linked directly to H(+) channel function in lung epithelium. The H(+) channel is activated by strong membrane depolarization, intracellular acidity, or extracellular alkalinity. Early on it was noted that these are surprising physiological channel characteristics when considering that lung epithelial cells have rather stable membrane potentials and a well pH-buffered intracellular milieu. This raised the question under which conditions the H(+) channel is active in lung epithelium and what is its physiological function there. Current understanding of the HVCN1 H(+) channel in lung epithelial acid secretion, its activation by an alkaline mucosal extracellular pH, and its role in the regulation of the mucosal pH of the lung has resulted in a model of mucosal pH regulation based on the parallel function of the HVCN1 H(+) channel and the CFTR HCO(3) (-) channel, which suggests that HVCN1 is a critical factor that maintains a neutral surface pH in the lung.

Studying Potassium Conductance in Isolated Mouse Heart Using Thallium Flux Assay

Inward rectifier potassium channels IK1 underlie major resting ionic conductance in the heart. A wealth of research on IK1 has been conducted on isolated cells using voltage-clamp techniques but studying IK1 in intact hearts is limited to ion flux assays. Additionally, in the past, flux studies were primarily performed in beating hearts thus eliminating the possibility of measuring any resting conductance. We have implemented a novel approach based on recently discovered thallium-dependent fluorescence of a low-affinity calcium-sensititive dye (BTC). Langendorff-perfused isolated mouse hearts were (i) loaded with membrane permeant BTC and (ii) stopped using verapamil and lidocaine, in order to achieve stable hyperpolarized membrane potential across the heart (thus silencing voltage-dependent potassium conductances). (iii) 1 mM thallium was then applied and (iv) fluorescence of BTC captured using a camera. In all hearts the time course of the relative increase in fluorescence, ΔF, was characterized by (1) well-defined fast (tau∼50÷150 sec) and (2) highly variable slow components. The total initial rate of ΔF was significantly increased in transgenic hearts with upregulated IK1 (up to 2-fold vs control). The initial rates of both components of ΔF were strongly IK1 dependent. Both, transgenic down regulation of IK1 and blockade of IK1 with Ba2+ ions led to a ∼10-20% decrease in the rate of ΔF (not statistically significant) suggesting other major pathways for thallium influx (e.g. Na+/K+ ATPase). Confocal microscopy of isolated ventricular myocytes revealed significant accumulation of BTC in the mitochondria consistent with the hypothesis that the slow component of ΔF reflects mitochondrial uptake of thallium. Results of this study show the possibility of studying sarcolemmal and mitochondrial K+ conductances including IK1 and other pathways (KATP, Na+/K+ ATPase, Na+/K+/CL- symporter) in intact isolated hearts with high temporal resolution.

Membrane potential and charge density of hollow-fiber dialysis membranes

Abstract Membrane charge affects diffusive permeability for ions. Hence, evaluation of membrane charge density is necessary to clarify diffusive permeability for ions. The objective of the present study is to measure stable and reproducible membrane potentials with hollow-fiber membranes and to evaluate the electrostatic characteristics of dialysis membrane by applying membrane potential measurement method for flat membrane to hollow-fiber membranes. Membrane charge density was calculated from data on the membrane potential. Values for membrane charge density obtained as electrostatic characteristics of AM-UP150 (regenerated cellulose), KF-15C (ethylenevinylalcohol copolymer), PAN-150SF (polyacrylonitrile), AM-BC150X (polyethylene glycol-modified regenerated cellulose), FB-150U (cellulose triacetate), APS-150E (polysulfone), FLX-15GW (polyester–polymer alloy) and BLF-10GW (diethylaminoethyl-modified regenerated cellulose) were −0.9, −0.9, −0.7, −0.5, −0.1, +0.1, +0.35 and +3.1 mol/m 3 , respectively. This method allows determination of the membrane charge density of hollow-fiber dialysis membrane.

Growth-dependent modulation of capacitative calcium entry in normal rat kidney fibroblasts

Normal rat kidney (NRK) fibroblasts have electrophysiological properties and intracellular calcium dynamics that are dependent upon their growth stage. In the present study we show that this differential behavior coincides with a differential calcium entry that can be either capacitative or non-capacitative. Confluent cells made quiescent by serum deprivation, which have a stable membrane potential near − 70 mV and do not show spontaneous intracellular calcium oscillations, primarily exhibit the capacitative mechanism for calcium entry, also called store-operated calcium entry (SOCE). When the quiescent cells are grown to density-arrest in the presence of EGF as the sole polypeptide growth factor, these cells characteristically fire spontaneously repetitive calcium action potentials, which propagate throughout the whole monolayer and are accompanied by intracellular calcium transients. These density-arrested cells appear to exhibit in addition to SOCE also receptor-operated calcium entry (ROCE) as a mechanism for calcium entry. Furthermore we show that, in contrast to earlier studies, the employed SOCs and ROCs are permeable for both calcium and strontium ions. We examined the expression of the canonical transient receptor potential channels (Trpcs) that may be involved in SOCE and ROCE. We show that NRK fibroblasts express the genes encoding Trpc1, Trpc5 and Trpc6, and that the levels of their expression are dependent upon the growth stage of the cells. In addition we examined the growth stage dependent expression of the genes encoding Orai1 and Stim1, two proteins that have recently been shown to be involved in SOCE. Our results suggest that the differential expression of Trpc5, Trpc6, Orai1 and Stim1 in quiescent and density-arrested NRK fibroblasts is responsible for the difference in regulation of calcium entry between these cells. Finally, we show that inhibition or potentiation of SOCE and ROCE by pharmacological agents has profound effects on calcium dynamics in NRK fibroblasts.

UP States Protect Ongoing Cortical Activity from Thalamic Inputs

Cortical neurons in vitro and in vivo fluctuate spontaneously between two stable membrane potentials: a depolarized UP state and a hyperpolarized DOWN state. UP states temporally correspond with multineuronal firing sequences which may be important for information processing. To examine how thalamic inputs interact with ongoing cortical UP state activity, we used calcium imaging and targeted whole-cell recordings of activated neurons in thalamocortical slices of mouse somatosensory cortex. Whereas thalamic stimulation during DOWN states generated multineuronal, synchronized UP states, identical stimulation during UP states had no effect on the subthreshold membrane dynamics of the vast majority of cells or on ongoing multineuronal temporal patterns. Both thalamocortical and corticocortical PSPs were significantly reduced and neuronal input resistance was significantly decreased during cortical UP states – mechanistically consistent with UP state insensitivity. Our results demonstrate that cortical dynamics during UP states are insensitive to thalamic inputs.